Figure 1
ME2 depletion inhibits proliferation.
(a), Knockdown of ME2 in pools of A549 with three independent ME2 shRNA lentiviruses. The full-length blots are presented in Supplemental Figure S7. (b), Cell proliferation with ME2 knockdown as described in “a”. (c), ME2 knockdown in single clones. The full-length blots are presented in Supplemental Figure S8. (d), Cell proliferation of single clones with ME2 knockdown as described in “c”. (e), Knockdown ME2 in H1650 cells. The full-length blots are presented in Supplemental Figure S9. (f), Cell proliferation with ME2 knockdown as described in “e”. (g), Knockdown of ME2 in A549 inhibits colony formation. (h), Fractional changes in 13C mass isotopologues (differing in the number of 13C atoms) of phosphatidylcholines (PC) in ME2 knockdown cells; m0: all 12C, m3: 13C3-PC (13C labeling in glycerol backbone only), m(even): 13Ceven#-PC (13C labeling in fatty acyl chains only), m(odd > 3): 13Codd#>3-PC (13C labeling in fatty acyl chains plus glycerol backbone). (i), Time courses of valine consumption, as determined from 1H NMR data. Metabolite data were expressed mean ± SEM, n = 2. (j), The effect of glutamine supplementation on ME2 depletion induced cell death. (k), Pyruvate rescues ME2 knockdown induced cell death. (l) and (m), The effect of ME2 knockdown on de novo synthesis of PC is greater with 13C6-Glc than with 13C5,1 5N2-Gln as tracer. (n), Inhibition of 13C3-citrate synthesis from 13C6-Glc by ME2 knockdown. (o), 13C atom-resolved tracing from 13C6-Glc through glycolysis, the Krebs cycle without or with input of pyruvate carboxylation and biosynthesis of fatty acyl chains with even number of 13C (13C2n) in lipids via citrate; respectively •,•: 13C atoms from the first turn of Krebs cycle without or with pyruvate carboxylation; solid and dashed arrows: single and multiple-step reactions; single and double-headed arrows: irreversible and reversible reactions; PDH: pyruvate dehydrogenase; PCB: pyruvate carboxylase. (p) and (q), Effects of ME2 knockdown on the levels and fractional distribution of 13C isotopologues of malate; red and green rectangles depict 13C2- (m2) and 13C3-malate (m3) species as markers of the first turn of the Krebs cycle without and with pyruvate carboxylation input, respectively. Metabolite data were expressed mean ± SEM, n = 2.
